Rapid Detections Of Pseudomoas Aeruginosa By Loop-mediated Isothermal Amplification

Pseudomonas aeruginosa bacteria as found in nature,is one of the pathogens commol/Lon surgical hospital.Clinical phenomenon indicates that P.aeruginosa infection in a wound form green pus,and induce sepsis,respiratory infections and urinary tract infections.Sepsis occurs mainly in the human extensive burns and cardiac surgery,patients often accompanied by symptoms such as shock or blood clotting can occur in patients with necrotizing pustule on the skin,usually in the patient’s blood vessel-forming bacteria have plug;the most commol/Lon respiratory tract infections is induced by mechanical respiratory ventilator-associated pneumonia;amputees because of catheter placement induced urinary tract infections,long-term use of antimicrobial treatment will also induce patients with Pseudomonas infections.Pseudomonas aeruginosa infection commol/Lon in skin damage,damp body parts,for example,between the baby’s umbilical cord around parts of the body as well as burn wet toes like.Lower incidence of normal human body,when people mention receive external factors can lead to lowered immol/Lunity,the bacteria can induce severe or fatal infections.Objective:To establish a rapid and sensitive method of loop-mediated isothermal amplification(LAMP)for detecting Pseudomonas aeruginosa(P.aeruginosa).Methods:The LAMP primers were designed according to the gbca gene of P.aeruginosa.A positive reaction was indicated by a color change before and after the reaction,which was verified by agarose gel electrophoresis.The sensitivity and specificity of the detection method were evaluated,and were compared with those of conventional PCR.Results:In this study,Pseudomonas aeruginosa in gbca design LAMP sequence of a gene,the establishment of a LAMP detection of Pseudomonas aeruginosa.Compared with conventional PCR,LAMP does not require the purification of DNA from the sample out of the application can be more rapidly detected clinical samples.Undeniably,LAMP technology also has some shortcomings,such as with respect to the terms of PCR,LAMP technology requires four primers were designed,its design requirements are relatively high;At the same time,once the non-specific amplification,the reaction will be intuitive gradient strip,misleading sample identification;for more sensitive LAMP characteristic experiment relatively easily contaminated.In order to examine more intuitive LAMP method established for the application of the identification of Pseudomonas aeruginosa,the study also added to the reaction system in a color change HNB dye determination LAMP technology to detect Pseudomonas aeruginosa gbca gene,and the method the sensitivity and specificity were investigated.From the experimental results can be seen: Results(1)color change and gel electrophoresis showed that only Pseudomonas aeruginosa gbca gene under LAMP amplification reaction method occurred,the other did not happen LAMP amplification negative bacteria,indicating that the specific measures were better;(2)LAMP amplification showed consistent results dye set gel electrophoresis,can successfully detect 8.7pg / ul concentration against Pseudomonas aeruginosa total DNA,and PCR method can detect only total DNA to a concentration of 87 pg/ul Pseudomonas aeruginosa,indicating LAMP detection sensitivity of PCR is about 10 times.While using PCR and LAMP parallel detection of clinically collected specimens of 21 parts by Pseudomonas aeruginosa bacteria,and 10 negative control,LAMP and PCR results showed the detection rate is very(21/21),two methods were not false positive event occurs.Conclusions:The established LAMP method in this study enables rapid,sensitive and specific detection of P.aeruginosa,and can be wide-used in medical institutions.